use of this formula is best shown with a worked example:
Dilution n colony count/plate total C x cfu ml^1 a
10 ^43 63, 74, 61 198 66 6.6 105
10 ^53 5, 11, 9 25 8.3 8.3 105
aCalculated from a single dilution
Weighted mean¼ð 198 þ 25 Þ
ð 3 þ 0 : 3 Þ1
0 : 1
1
10 ^4
¼ 6 : 8 105
Traditional plate counts are expensive in Petri dishes and agar media,
especially if adequate replication is carried out, and the Miles and Misra
drop count and spiral plater have been developed to reduce costs. In the
Miles–Misra technique materials are conserved by culturing a smaller
volume of each dilution, usually 20ml. This way a number of dilutions
can be grown on a single plate by dividing it into sectors each repre-
senting a different dilution. The spiral plater employs a mechanical
system which dispenses 50ml of a liquid sample as a spiral track on the
surface of an agar plate. The system is engineered so that most of the
sample is deposited near the centre of the plate with a decreasing volume
applied towards the edge. This produces an effect equivalent to a
dilution of the sample by a factor of 10^3 on a single plate, thus
producing a two-thirds’ saving on materials as well as saving the time
required in preparing and plating extra dilutions. After incubation,
colonies are counted using a specially designed grid which relates plate
area to the volume applied, thus enabling the count to be determined.
The system is not suitable for all food samples though, as particulate
material can block the hollow stylus through which the sample is
applied.
The limit in sensitivity of the traditional plate count arises from the
small volumes used and clearly, the sensitivity can be increased by
increasing the volume size and the number of replicate counts or plates.
It may be possible to filter a larger volume through a membrane, which
retains the viable organisms, and then lay the membrane onto an
appropriate medium.
In all of these methods of enumeration it is essential to appreciate the
statistical background to sampling and to recognize that extrapolations
from colony counts depend on several assumptions that may not be
justified. Thus a colony may not be derived from a single micro-organ-
ism, but from a clump of micro-organisms, and the material being
examined may not be homogeneous so that the subsample actually
studied is not representative of the whole.
Chapter 10 379